Association of Polyphenol Oxidase Activities with Molecular Markers and Yellow Rust Resistance in Diverse Bread Wheat Genotypes

Research Article

Austin J Anal Pharm Chem. 2021; 8(2): 1134.

Association of Polyphenol Oxidase Activities with Molecular Markers and Yellow Rust Resistance in Diverse Bread Wheat Genotypes

Asghar MN1#*, Bajwa AA2#, Ali A3, Muhammad A2, Sami A2 and Saqlan Naqvi SM2

1Department of Medical Biology, University of Quebec, Trois Riveres, Canada

2University Institute of Biochemistry and Biotechnology, PMAS-UAAR Rawalpindi, Pakistan

3Centre for Plant Sciences and Biodiversity, University of Swat, KPK, Pakistan

#Equal Contributed to this Work

*Corresponding author: Muhammad Nadeem Asghar, Department of Medical Biology, University of Quebec, Trois Riveres, Canada

Received: July 31, 2021; Accepted: August 27, 2021; Published: September 03, 2021

Abstract

Polyphenol Oxidase (PPO) catalyses the undesirable browning of wheat products which is of significant concern in consumer acceptance perspectives. Another important yield-limiting cause for wheat crops is wheat rust (e.g., yellow rust), a source of great economic loss worldwide. The purpose of the current research was to screen conventional and synthetic bread wheat genotypes for their PPO activity and yellow rust resistance. Different genotypes differed significantly in total PPO activity and in their activities against different substrates (L-DOPA and Catechol). The synthetically derived bread wheat genotypes 1-279, showed the lowest (39.2 units/min/g) cumulative PPO activity. Ten genotypes each with the highest and lowest PPO activities were selected for testing their association with seven reported molecular markers. Intriguingly the PPO markers reported in literature could not clearly differentiate between contrasting cultivars. Association with yellow rust resistance was also investigated. Interestingly, the rust-resistant genotypes, including 1-263, Ch-43, 1-57 and Emat (all synthetic-derived), exhibited low PPO activity. The current study underpins that there is a need to search for more reliable PPO markers and to further validate association of low PPO activity with yellow rust.

Keywords: Browning; Polyphenol oxidase activity; PPO; Yellow rust resistance; Synthetic-derived wheat

Introduction

Wheat is the second major staple crop of the world. Improved wheat products will ensure continuing international cooperativeness and sustainability of wheat industry. Routinely used wheat varieties are Durums; which are allotetraploid (AABB; 2n = 4x = 28) and common wheats; which are allohexaploids (AABBDD; 2n = 6x = 42) [1]. Resistance to stress and disease is of major concern to wheat breeders. Synthetic hexaploid wheat is better adapted to high temperature, drought, salinity, waterlogging and soil micronutrient imbalances. However, the most important hurdle in the up-gradation of the wheat industry is browning wheat-derived products that affect their market value. The leading cause of this undesirable browning is Polyphenoloxidases (PPO), therefore considerable efforts are required to reduce levels of PPO for prevention of economic loss [2,3]. There are other methods and combinations to inhibit the browning of wheat [4], and further tools are searched to limit the browning [5].

PPO is a nuclear-encoded copper-containing enzyme that is distributed from bacteria to mammals. PPO is enzymatically complex as it catalyzes the hydroxylation of monophenols to o-diphenols and oxidation of o-diphenols to o-quinones. The quionones covalently modify amines, thiols and phenolics forming colored polymers, which are responsible for browning of food products. PPO was first reported in wheat bran [6] and has been extensively studied in other plants [7]. PPO also has some wide applications in the industry such as its use as a biosensor in industrial waste waters [8].

PPO has been found in multiple molecular forms (isozymes) in various plants, at least 12 isoforms of PPO have been identified in a wheat kernel during different stages of development [9,10]. The existence of PPO isoforms has been attributed to modification of enzyme during the isolation process or it could also occur during processing of nuclear coded proteins or due to differential expression of different members of a gene family [11]. Jukanti et al. [12] hypothesized that the PPO gene in wheat has evolved by gene duplication into a multigene family. The total PPO activity of any plant is difficult to access because PPO exists in both latent and active forms, PPO activity is also quite variable in the presence of different phenolic substrates, it depends upon the nature of the side chain, number of hydroxyl groups and its position in benzene ring of substrate [11]. Due to its broad substrate specificities, PPO has an array of names such as phenol oxidase, monophenol oxidase and tyrosinase.

PPO activity in wheat grain is influenced by environment and genotype [13,14]. Location of PPO, analyzed through QTL analysis, showed that the chromosomes 2A, 2B, 2D, 3D and 6B contains PPO genes [15-18]. 2D chromosome was found to be associated with elevated levels of PPO activity in kernels of hexaploid wheat [19] as compared to tetraploid wheat, which lacks the genome-D and have high activity associated with the long arm of chromosome 2A [17,18]. PPO enzyme is located in the aleuron layer of wheat, which is removed during milling; however, the contamination by bran layer is enough to cause enzymatic browning [20]. Development of wheat varieties having low PPO activity is one of the priorities today because discolored food products are not preferred by consumers. PPO activity is a physiological and biochemical trait so it cannot be accessed based on morphological characteristics. However, the use of molecular markers linked to major QTLs of PPO can facilitate our search for low PPO varieties.

Another important yield-limiting cause for wheat crops that occur throughout the world is wheat rust. Three wheat rust diseases, stem rust, leaf rust and stripe (yellow) rust are a source of great economic loss worldwide [21,22]. Genome wide association studies are carried out in India, Kenya, Mexico and Egypt identified QTLs associated with Yellow rust [23,24]. Severe infection of stripe rust is caused by Puccina striiformis with yield losses in susceptible cultivars of up to sixty percent [25]. It is referred to as yellow or stripe rust because of the powdery yellow-orange masses of pustules arranged in stripes along the venation of leaf during spring and summer [26] giving characteristic striped appearance. Breeding for resistance is the most economical, environment friendly and the effective way than the use of fungicides for controlling the disease. Fungal (Alternaria triticinia) inoculation on resistant wheat cultivar is associated with the production of more PPO activity and this increase in enzyme concentration is well documented [27]. Several reports suggest a positive correlation between PPO activity and disease resistance [28,29]. Fungal infection on a range of tomato cultivar, from highly resistant to highly susceptible, suggested a considerable positive correlation between resistance, PPO activity and accumulation of phenol. Inoculated resistant cultivars showed higher PPO activity and accumulation of phenol than the inoculated susceptible cultivar. A higher PPO activity and phenol content were found in mature leaves of inoculated resistant cultivar. Disease index, PPO activity and phenol concentration were found to be directly correlated in the tissue [29].

The present study was carried out to screen the synthetic-derived bread wheat genotypes on the basis of their PPO activity; discover the correlation/association with resistant/susceptible cultivars to stripe rust and to check the association of molecular markers with the PPO activity in selected wheat lines.

Materials and Methods

The experimental plant materials consisted of wheat diverse genotypes comprising of total of 95 synthetic derived lines and check cultivars. Synthetic derived wheats were produced earlier from crosses of primary synthetic hexaploid wheats with advanced and improved cultivars and was obtained for research purpose from international maize and wheat improvement center (CIMMYT) through wheat wide crosses and cytogenetics program at National Agriculture Research Center (NARC), Islamabad, Pakistan. Their PPO activities were determined on L-DOPA and Catechol substrates.

Measurement of PPO activity with catechol and L-DOPA

The PPO activity with L-DOPA and catechol substrate was determined according to the method described by Fuerst et al. [30] with little modification. Experiments were performed in triplicate for each variety and substrate. Substrates were prepared in a buffer of 50mM³-(N-morpholino) propane sulfonic acid (MOPS) and pH was adjusted to 6.5. In each test tube containing seeds, 1.5mL (10mM) of phenolic substrate was added and tubes were incubated at 25°C for about 2 hours with rotation at 250rpm. Following incubation, absorption was measured through spectrophotometer and the change in absorbance was compared with a control containing only substrate. Absorbance was measured at 410nm and 475nm for catechol and L-DOPA respectively. One unit of PPO is defined as a change in absorbance by 10-3/min/g seed. The PPO activity with all the substrates was calculated as; Enzyme activity=ΔOD/min/g seed.

Selection of seed varieties for molecular markers study

The top ten high and low PPO activity varieties were selected to investigate further their association with molecular markers and rust resistance (Table 1 and 2). Selected varieties were germinated in autoclaved Petri dishes. Seeds were placed on filter paper under moist conditions. Genomic DNA was extracted from leaves after a week by the CTAB method described by Richards.